1. Field of the Invention
This invention relates to a hybridoma cell line and monoclonal antibody produced therefrom which may be used to detect potato, tomato, and/or eggplant glycoalkaloids.
2. Description of the Prior Art
Alkaloids are potentially toxic nitrogen-containing secondary plant metabolites found in numerous plant species, including potatoes and tomatoes (Friedman, 1992, J. Agric. Food Chem., 40:419-423, and ACS Symp. Ser., No. 406, 429-462). The two major glycoalkaloids in commercial potatoes (Solanum tuberosum), .alpha.-chaconine and .alpha.-solanine, are both glycosylated derivatives (triosides) of the aglycon solanidine. Wild potatoes (Solanum chacoense) and eggplants (Solanum melongena) produce the glycoalkaloid solasonine. The major glycoalkaloid in tomatoes (Lycopersicon esculentum), .alpha.-tomatine, is the glycosylated derivative of the aglycon tomatidine.
The potato glycoalkaloids are thought to function as a defense against insects and other pests (Norris, 1986, In Chemistry of Plant Protection, Haug and Hoffman eds., Springer Verlag, Berlin, Germany, pp. 97-146). Because wild potatoes often contain higher glycoalkaloid levels than commercial varieties, they have been used by plant breeders attempting to generate improved cultivars. Such cultivars, however, can have glycoalkaloid levels above 20 mg/100 g of tuber, the generally accepted cutoff level between safe and unsafe potatoes (Slanina, 1990, Food Chem., 28:759-761; and Van Gelder, 1991, In Handbook of Natural Toxins, Vol. 6, Toxicology of Plant and Fungal Compounds, Keeler and Tu, eds., Marcel Dekker Inc., New York, pp. 101-134). This guideline limiting the glycoalkaloid content of new potato cultivars has been recommended because of the potential human toxicity of these compounds, including reported deaths (McMillan and Thompson, 1979, Quaterly Journal of Medicine, 48:227-243; and Morris and Lee, 1984, Food Technology in Australia, 36:118-124).
Detection of glycoalkaloids in potato and tomato plants is of interest because of the toxic nature of these compounds. Traditional methods to detect these plant constituents are often complicated and time consuming, and they rely upon the use of large amounts of organic solvents. In addition, the reported methods are not readily field portable, and most require sophisticated and expensive equipment and trained personnel to run the assays and interpret the results.
Methodologies that have been disclosed for the analysis of glycoalkaloids and related compounds include gas chromatography (Herb et al., 1975, J. Agric. Food Chem., 23:520-523; Lawson et al., 1992, J. Agric. Food Chem., 40:2186-2191; and Van Gelder et al., 1989, J. Chromatogr., 482:13-22), and high-performance liquid chromatography (Bushway, 1982, J. Liq. Chromatogr., 5:1313-1322; Bushway et al., 1979, J. Chromatogr., 178:533-541; and 1986, J. Agric. Food Chem., 34:277-279; Carman et al., 1986, J. Agric. Food Chem., 34:279-282; Friedman and Levin, 1992, J. Agric. Food Chem., 40:2157-2163; Morris and Lee, 1981, J. Chromatogr., 219:403-410; and Osman and Sinder, 1989, J. Chromatogr., 479:189-193).
Immunoassays for potato glycoalkaloids have been described. Ward et al. (1988, Food Addit. Contam., 5:621-627) and Plhak and Sporns, 1992, J. Agric. Food Chem., 40:2533-2544) described polyclonal antisera raised using immunogens produced by complex conjugation strategies. These immunogens were produced by first cleaving the carbohydrate side chain by periodate to aldehyde groups, followed by Schiff's base formation with BSA and reduction of the Schiff's bases by sodium borohydride. Other immunoassays for potato glycoalkaloids have also been described by Morgan et al. (1983, J. Sci. Food Agric., 34:593-598; and 1985, In Immunoassays in Food Analysis, Morris and Clifford eds., Elsivier Applied Science Publishers, London, UK, pp. 187-195), Plhak and Sporns (1994, Am. Potato J., 71:297-313), Thomson and Sporns (1995, J. Agric. Food Chem., 43:254-260) and Vallejo and Ercegovich (1978, In Methods and Standards for Environmental Measurements, Publication 519, National Bureau of Standards, Washington, D.C., pp. 333-340). Barbour et al. (1991, Rev. Pesticide Toxicol., 1:289-303) also described an immunoassay for the tomato glycoalkaloid, .alpha.-tomatine. However, the above studies all used the glycoalkaloid itself, or a modified glycoalkaloid as immunogen, linking to the carrier protein via a modification in the sugar. Generally, these immunogens have not resulted in antibodies of sufficiently high affinity to be suitable for use in commercial assays.